1. Field of the Invention
The invention relates to the treatment of diseases caused by viral infection. More precisely, the invention relates to a method of prophylactic and/or therapeutic treatment of a mammal for a disease. The disease may be caused by viral infection in most tissues or cell types. For example, the disease may be in muscle tissues, neural cells, or endocrine glands caused by a Ljungan virus infection. The invention also relates to the use of an antiviral compound effective against a Ljungan virus for the preparation of a medicament for the treatment of a disease. The disease may be caused by viral infection in most tissues or cell types. For example, the disease may be in muscle tissues, neural cells, or endocrine glands caused by a Ljungan virus infection.
2. Background Art
Rodents are well-known reservoirs and vectors for viruses causing disease in humans. Puumala virus causing Nephropathia Epidemica (Myhrman, Nordisk Medicinsk Tidskrift, 7, 739-794, 1934; and Niklasson et al., Lancet, 1, 1012-3, 1984) is one example of an important human pathogen carried by bank voles. It has been demonstrated that the incidence rate of human Nephropathia Epidemica correlates with the vole population density during the previous year (Niklasson et al., Am. J. Trop. Med. Hyg., 53, 134-40, 1995). More recently, statistical evidence suggests that type 1 diabetes in humans also tracks the 3- to 4-year population density cycles of the bank vole with a similar time lag (Niklasson et al., Emerg. Infect. Dis., 4, 187-93, 1998). It was also observed that a high frequency of bank voles trapped in the wild and kept in the laboratory for studies of stereotypic behavior (Schoenecker et al., Appl. Anim. Behav. Sci., 68, 349-357, 2000) develop polydipsia and glucosuria at a high frequency.
Ljungan virus, belonging to the Picornavirus family, is carried by small rodents and causes diseases in other animals, including humans. The first three isolates were disclosed in the International Patent Application WO 98/11133 and the partial sequence of each was also comprised therein. The full sequences of these three Ljungan viruses have recently been published. (Johnsson S. et al., Journal of Virology, September 2002, p. 8920-8930)
Ljungan virus is a virus carried by small rodents. Ljungan virus belongs to the Picornavirus family. Ljungan virus is serologically and genetically distantly related to other members of the Picornavirus family. Ljungan virus will most likely form a new genus in the Picornavirus family.
Genetically, the Ljungan virus genome and the polyprotein encoded by them exhibit several exceptional features, such as the absence of a predicted maturation cleavage of VP0, a conserved sequence determinant in VP0 that is typically found in VP1 of other Picornaviruses, and a cluster of two unrelated 2A proteins. The 2A1 protein is related to the 2A protein of cardio, erbo and aphthoviruses and the 2A2 protein is related to the 2A protein of parechoviruses, kobuviruses and avian encephalomyelitis virus. (A. M. Lindberg and Susanna Johansson, Virus Research 85 (2002) 61-70).
Ljungan virus is characterized by a chronic or long lasting infection in its rodent host and reservoir. Ljungan virus can replicate and cause disease in a very broad host spectrum of animal species as well as in humans. Ljungan virus infects these different species of animals as well as humans and the infection does often result in a long lasting or chronic infection. Ljungan virus replicates in a wide variety of tissue culture cells giving a chronic infection with discrete CPE (cytopathogenic effect) and low viral output (in the order of 1000-100 000 viral particles per ml supernatant).
Data generated by virus cultivation under laboratory conditions show that Ljungan virus grows/replicates in a number of cell lines that originate from different tissues and different species, e.g. Vero monkey kidney; Vero E6 monkey kidney; MA-104 monkey kidney; CV-1 monkey kidney; GMK monkey kidney; A-549 human lung; Hela human cervical tissue; BHK 21 hamster kidney; RD human muscle; and L-cells mouse skin.
In living animals and humans Ljungan virus replicates in muscle tissue including heart tissue, in neural cell including the brain, in endocrine glands including the beta cells of the pancreas, the thyroid gland, the supra renal gland.
Data generated by detection of virus by Ljungan virus specific immunohistochemistry test, thin section electron microscopy and by PCR in humans, bank voles, lemmings, laboratory mice rabbits, guinea pigs, arctic foxes, and moose show that Ljungan virus has been found in endocrine and exocrine pancreas tissue, in endothelial cells of vessels, cells in the brain (including nerve tissue), cells of the liver, cells of the placenta and the umbilical cord, muscle tissue, heart tissue, tissue of the thyroid gland. The conclusion is that Ljungan virus can grow in most cell types of the body and therefore infect all organs of the body.
The only group of viruses that partly interferes with the above definition of Ljungan viruses are viruses in the cardio virus genus. There are some similarities among Ljungan viruses and viruses in the cardio virus genus. For example, cardioviruses belong to the picornavirus family, cardiovirus have rodents as their natural reservoir, cardioviruses can cause disease in a wide variety of animal species, and cardiovirus can infect and cause disease in the same organs as Ljungan virus. There are also some differences between Ljungan viruses and viruses in the cardiovirus genus. For example, cardiovirus and Ljungan virus are genetically distantly related, the double 2 A of Ljungan virus is absent in cardiovirus, cardiovirus is not related to Ljungan virus by serology, cardiovirus cause an acute disease (not long lasting or chronic) when it affects its non-rodent victims, cardiovirus is easy to cultivate in tissue culture without adaptation while Ljungan virus is often impossible to cultivate without blind passage in tissue culture or first passage in suckling mice and after several passages in suckling mice adaptation in tissue culture, and cardioviruses do not infect humans (only rare case reports in the literature).
New variants of Ljungan virus can be found in different continents. They will all be carried by small rodents and they will all cause the same disease syndromes in humans. The source for virus isolation/discovery can be selected/identified in different ways. For example, a wild rodent such as a mouse, rat or a vole with signs and symptoms similar to the diseases linked with Ljungan virus in humans (e.g. diabetes or myocarditis) can be found. Additionally, a screen of a large numbers of wild rodents by PCR using several different primer combinations targeting the conserved region of the Ljungan virus genome can be done. Further, a screen of a large number of wild rodents using specific antiserum can be performed. Antisera are collected from patients with the disease in humans linked with Ljungan virus that are living in the same geographic area as the rodents. Ljungan virus infected rodents are identified by immunostaining (e.g. immunohistochemistry) of formalin fixed organs. A portion of the same organ that is tested by immunohistochemistry is kept without fixation in a minus 70° C. freezer. The unfixed material is used for virus isolation if the immunohistochemistry turns out to be positive.
Tissue, from which virus isolation attempt will be made, is grinded and diluted in sterile saline or PBS. One-day old suckling mice are injected with 2-4 microliters of the tissue suspension intracerebrally. When suckling mice are used for virus isolation, in general all the mice die within a week of inoculation if a virus is present. However, Ljungan virus is different in that you have to wait 10 days to 3 weeks before signs of symptoms in the baby mice develop. Signs and symptoms are very discrete such as slow weight increase and altered mobility. Only 5-10% of the animals develop symptoms. This is very unusual and would in most cases result in a negative interpretation of the isolation attempt. Only the brain tissue from suckling mice with signs and symptoms are used for passage in new one-day old suckling mice.
When passed, the brains from sick suckling mice are grinded and diluted in sterile saline or PBS. One-day old suckling mice are injected with 2-4 microliters of the tissue suspension intracerebrally. Several such passages may be necessary before disease develops earlier (8-12 days) and in the majority of mice. After several passages in suckling mice Ljungan virus is inoculated into tissue culture such as Vero cells for amplification and identification.
Ljungan virus must be adapted to cell culture by passages of the cells. No or very discrete cytopathogenic effect is seen. The cells (not the tissue culture fluid) are passed weekly into new tissue culture bottles at a rate of 1 to 5. After 3-6 such blind passages the cells are stained using antibodies directed to the isolate. These antisera can be made by immunising adult mice with the suckling mouse brain suspension of a suspected isolate and/or by using human serum from patients with the disease caused by Ljungan virus living in the same geographic region as the animals used as source for virus isolation.
The ability to identify and associate an infectious agent with human disease is profoundly influenced by its biological characteristics. Infectious agents that are difficult to detect and cultivate increase the difficulties of linking the agent to disease. Additionally, zoonotic agents have low selective pressure for efficient growth in a human host, which can result in low pathogen concentrations, making viral identification difficult. Epidemiological observations on the vector often precede development of the diagnostic tools necessary for linking the agent to disease. In addition, incubation time, disease incidence and disease severity all influence the success of connecting a potential pathogen to human disease.
Hantavirus, which is a widely distributed viral infection with various rodent species serving as vectors, is a good example of the challenges involved in viral identification. In the human host, hantavirus manifests a range of within and between strain symptoms. Despite scientific attention drawn to the hemorrhagic fever characteristic of Old World hantavirus infection throughout much of the 20th century, and the fact that incubation time is short and the disease severe or lethal, it was only in 1978 for Asia and in 1984 for Europe that the viral agents were identified, and in 1993 that the New World lade of this pathogen recognized. The discovery of hantavirus illustrates the need to integrate epidemiology, vector biology and microbiology to identify the cause of such zoonotic disease.
It was recently proposed that lethal myocarditis and type 1 diabetes (T1D) in humans can be caused by one or more infectious agents carried by rodents, based on the association between rodent density and disease incidence. Based upon these observations, a search for an etiologic agent in small rodents was done. This resulted in the isolation of a novel picornavirus from the most prevalent mammal in northern Europe, the bank vole (Clethrionomys glareolus). This virus is called the “Ljungan virus” after the Valley in Västernorrland County in Sweden where it was first observed.
The first observation of LV-related diseases was recently made in bank voles, which showed that animals in captivity developed diabetes. Polyuria, polydipsia, glucosuria, elevated blood glucose levels, ketoacidosis and deaths were observed. Lysis of pancreatic islet beta cells and presence of GAD65-, IA-2 and insulin auto-antibodies suggested that the bank voles suffered from a T1D condition. Bank voles colonized for more than a decade were studied in more detail and it was found that approximately 20% of the animals developed an abnormal glucose tolerance test, accompanied by high serum insulin levels, increased insulin release from isolated islets, and a glucose oxidation rate consistent with type 2 diabetes (T2D) (Blixt, M., Niklasson, B. & Sandler, S. Characterization of β-cell function of pancreatic islets isolated from bank voles developing glucose intolerance/diabetes: an animal model showing features of both type 1 and type 2 diabetes mellitus). Moreover, total destruction of beta cells was also found in these colonized bank voles, similar to the pathology observed in recently trapped voles from cyclic populations in northern Sweden. These animals thus go through a T2D-like phase with viable beta cells, evidence of insulin resistance indicated by abnormal glucose tolerance tests and high serum insulin levels with progression to T1D with total destruction of beta cells.
It has been recently found that a high proportion of live-trapped bank voles, grey sided voles (C. rufocanus), field voles (Microtus agrestis) and Norwegian lemmings (Lemmus lemmus) suffer from diabetes and myocarditis when tested at peak densities in cyclic populations in northern Scandinavia (Niklasson B, Nyholm E, Feinstein R E, Samsioe S, Lernmark Å, Hörnfeldt B. Virus-induced diabetes and myocarditis in voles and lemmings at cyclic peak densities). These diseases have been associated with LV infection in wild rodents, and compelling evidence suggests that infected animals develop disease when subjected to stress. LV-infected laboratory mice infected during the first weeks of life develop encephalitis, myocarditis and pancreatitis, followed by diabetes, as measured by abnormal glucose tolerance test 10-15 weeks after infection (Niklasson B, Nyholm E, Feinstein R E, Samsioe S, Lernmark Å, Hörnfeldt B. Virus-induced diabetes and myocarditis in voles and lemmings at cyclic peak densities). By inducing these diseases under controlled laboratory conditions Koch's postulates proving causality were fulfilled. The frequency of diabetes varied from approximately 80% of the male mice infected during the first three days of life to 30% of the male mice infected during the second week of life. Very few female mice developed diabetes.